Norepinephrine Compositions and Methods Therefor

ABSTRACT

The inventive subject matter is directed to compositions and methods for ready-to-inject norepinephrine compositions with improved stability. Most preferably, compositions presented herein are substantially antioxidant free and exhibit less than 10% isomerization of R-nor epinephrine and exhibit less than 5% degradation of total norepinephrine.

This application is a divisional application of our copending USprovisional application with Ser. No. 15/883,798, which was filed Jan.30, 2018, which claims priority to US provisional application with Ser.No. 62/452,220, which was filed Jan. 30, 2017.

FIELD OF THE INVENTION

The field of the invention is pharmaceutical compositions comprisingnorepinephrine, especially as it relates to storage stable,ready-to-inject, antioxidant free compositions, and method ofmanufacturing such compositions.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Norepinephrine is often used during CPR (cardio-pulmonaryresuscitation), and in the treatment of cardiac arrest and profoundhypotension. Norepinephrine is also used for blood pressure control incertain acute hypotensive states, including for example sympathectomy,poliomyelitis, pheochromocytomectomy, spinal anesthesia, myocardialinfarction, blood transfusion, and septicemia.

Currently, norepinephrine is marketed as Levophed®, which is aconcentrated 4 mg per 4 mL norepinephrine bitartrate formulation to beadministered by intravenous infusion following dilution with dextrose ordextrose and sodium chloride injection. Norepinephrine is also marketedby Baxter which supplies as a norepinephrine concentrate that is free ofsodium metabisulfite and packaged under nitrogen. Unfortunately, most,if not all diluted commercially available norepinephrine formulationslack storage and should therefore be discarded within one day afterreconstitution when stored at room temperature. Consequently, risk formicrobial contamination and dilution errors is present. In addition,Levophed also contains sodium metabisulphite as an antioxidant, andcarries a warning label that sulfite may cause allergic type reactionsincluding anaphylactic shock and life threating or less severe asthmaticepisodes in susceptible people. Table 1 depicts ingredients of currentlymarketed norepinephrine compositions.

TABLE 1 Composition of currently marketed Norepinephrine BitartrateProducts. Norepinephrine Levophed ® Bitartarate Ingredient (Hospira)(Baxter) Norepinephrine Bitartrate 1 mg/mL 1 mg/mL equivalent toNorepinephrine Base Sodium Chloride Isotonic Isotonic SodiumMetabisulphite 0.2 mg/mL — pH 3-4.5 3-4.5 Water for injection q.s. 1 mLq.s 1 mL

Stability of Levophed® and Norepinephrine bitartrate injection (Baxter),in normal saline solutions is presented in Table 2 and Table 3 wherenorepinephrine was diluted to a concentration of 16 μg/ml. Stability wasassessed in 250 ml saline at accelerated (i.e., 40±2° C. and 75±5%relative humidity, duration as indicated) and long term stability (i.e.,25±2° C. and 60±5% relative humidity, duration as indicated) storageconditions.

TABLE 2 Stability study of Levophed ® diluted in 0.9% Saline (Hospira)at 16 μg/mL. Storage Condition 25 ± 2° C./60 ± 5% RH 40 ± 2° C./75 ± 5%RH Time Point Initial 1 Month 2 Month 3 Month Initial 1 Month 2 Month 3Month Assay 97.3 98.9 97.9 91.9 98.8 96.5 80.2 71.9 Total 0.05 — 0.718.08 0.03 1.96 5.29 9.73 Impurities

TABLE 3 Stability study of Norepinephrine bitartrate injection [Baxter]diluted in 0.9% Saline (Hospira) at 16 μg/mL. Storage Condition 25 ± 2°C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2 Month 3Month Initial 1 Month 2 Month 3 Month Assay 99.9 99.7 97.0 92.2 99.491.5 82.9 77.6 Total 0.08 1.73 2.68 10.17 0.10 2.34 4.46 6.71 Impurities

As can be seen from the results, the norepinephrine at ready-to-injectconcentrations underwent significant degradation. Oxidative degradationcould possibly be reduced or even prevented by addition of effectiveamounts of sodium metabisulphite to the ready-to-inject norepinephrinesolution. However, the quantities of sodium metabisulphite that would beadministered by injection of 250 ml of the ready-to-inject solutionwould be substantial and detrimental to the patient. To avoid issuesassociated with sodium metabisulphite, efforts have been made to providenorepinephrine formulations with a non-sulfite anti-oxidant. For exampleUS 2016/0058715 teaches a ready-to-inject dosage form of norepinephrinethat uses butylated hydroxyl anisole as an anti-oxidant. While generallydeemed safe for topical and cosmetic use, butylated hydroxyl anisole wasshown to produce some renal and hepatic damage (e.g., Int J Toxicol.2002; 21 Suppl 2:19-94).

In other attempts to provide ready-to-administer norepinephrineformulations with increased storage stability and reduced risk of humanerror, the pH on the injectable solution was reduced to between 3.2 and3.6 with 40-200 μg/ml norepinephrine as is described in WO 2015/128418.While such formulations exhibited reduced degradation as compared tohigher pH formulations, significant discomfort can occur at theinjection site. Worse yet, at the pH used, norepinephrine isomerizedrelatively quickly from the active R (−) isomer to the inactive S (+)isomer. Isomerization is also encountered at exposure of norepinephrineto higher temperatures.

Therefore, there is a need for improved stable, low concentration,ready-to-inject and antioxidant free norepinephrine formulations, andmethods of manufacturing and storing the same.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to antioxidant freesterilizable/autoclavable ready-to-inject norepinephrine compositionshaving improved stability and a physiologically acceptable pH.

In one aspect of the inventive subject matter, the inventors contemplatea ready to ready-to-inject norepinephrine composition that comprises anaqueous acidic buffer having a pH range of between 3.7 and 4.3, whereinthe aqueous buffer further comprises a chelating agent and apharmaceutically acceptable salt. Most typically, the chelating agent ispresent in an amount of between 1 μg/ml and 100 μg/ml, and thepharmaceutically acceptable salt is present in an amount of between 0.6wt % and 1.2 wt %. Norepinephrine (typically enantiomerically pure(i.e., at least 98%) R-isomer) is dissolved at a concentration that issuitable for administration to a patient in need thereof. In furtherpreferred aspects, the ready-to-administer norepinephrine composition issubstantially free of antioxidants, and the ready-to-administernorepinephrine composition is formulated such that after storage over atleast three months equal or less than 10% of the R-isomer form willisomerize to the S-isomer and such that equal or less than 5% of thetotal norepinephrine will degrade to degradation products. As usedherein, reference to the term norepinephrine should be interpretedbroadly to include pharmaceutically acceptable salts and prodrugsthereof.

Therefore, the inventors also contemplate a method of preparing aready-to-inject norepinephrine composition that includes a step offormulating a liquid parenteral composition that contains in an aqueousacidic buffer norepinephrine as an R-isomer such that (a) theformulation exhibits less than 10% of isomerization of the R-isomer toan S-isomer after three months of storage of the liquid composition, and(b) the formulation exhibits equal or less than 5% degradation of totalnorepinephrine after three months of storage of the liquid composition.The aqueous acidic buffer will typically have a pH range of between 3.7and 4.3, and the aqueous buffer will further comprise a chelating agentand a pharmaceutically acceptable salt. In such methods, the totalnorepinephrine is present in the liquid parenteral composition at aconcentration of between 10 μg/ml and 100 μg/ml, and the ready-to-injectnorepinephrine composition is substantially free of antioxidants.

Viewed form a different perspective, the inventors also contemplate amethod of preparing a ready-to-inject norepinephrine composition thatincludes the steps of preparing an aqueous acidic buffer at a pH rangeof between 3.7 and 4.3, wherein the aqueous buffer also includes achelating agent and a pharmaceutically acceptable salt. Preferably, thechelating agent is present in an amount of between 1 μg/ml and 100μg/ml, and tonicity is adjusted with the pharmaceutically acceptablesalt (e.g., NaCl). In a further step, norepinephrine (preferablyenantiomerically pure R-isomer) is dissolved at a concentration suitablefor administration to a patient in need thereof, and theready-to-administer norepinephrine composition is formulated such thatafter storage over at least three months equal or less than 10% of theR-isomer form will isomerize to the S-isomer and such that equal or lessthan 5% of the total norepinephrine will degrade to degradationproducts. As before, it is generally preferred that theready-to-administer norepinephrine composition is substantially free ofantioxidants. In yet another step, the composition is autoclaved tosterility.

Most typically, but not necessarily, the aqueous acidic buffer is acitrate buffer and/or preferably has a concentration of between 5 mM and20 mM. Furthermore, preferred aqueous acidic buffers will have a pH ofbetween 3.8 and 4.2. With respect to the chelating agent it iscontemplated that such agents are a bicarboxylic acid (e.g., optionallyhydroxylated, tartrate), a tricarboxylic acid (e.g., aconitic acid,trimesic acid, citric acid), and/or an aminopolycarboxylic acid (e.g.,EDTA, EGTA, etc.), and that such chelating agents are present at lowconcentrations, preferably between 1 μg/ml and 10 μg/ml, or between 10μg/ml and 100 μg/ml. The norepinephrine is typically present at aconcentration of between 10 μg/ml and 100 μg/ml, for example, at aconcentration of 16 μg/ml (+/−10%), 32 μg/ml (+/−10%), or 64 μg/ml(+/−10%). Contemplated methods may also include a step of autoclavingthe compositions.

With respect to stability it is contemplated that the storage conditionis over at least three months at 40° C. and 75% (+/−5) relativehumidity, that equal or less than 6% of the R-isomer form will isomerizeto the S-isomer, and/or that equal or less than 3.5% of the totalnorepinephrine will degrade to degradation products.

Where desired, contemplated compositions have a dissolved oxygenconcentration of equal or less than 1 ppm (e.g., by formulating theliquid parenteral composition using deoxygenated water), and/or or bypackaging the composition together with a (preferably metal free) oxygenscavenger. Packaging may further make use of a container that isconfigured (e.g., aluminized or otherwise treated) to reducelight-mediated oxidation of the norepinephrine.

DETAILED DESCRIPTION OF THE INVENTION

The inventive subject matter is directed to stable aqueouspharmaceutical preparations of norepinephrine (and pharmaceuticallyacceptable salts thereof) in a ready-to-inject form that are sterile andpreferably substantially free of antioxidants. Most preferably,stability of such compositions is characterized by low (oxidative andphoto-induced) degradation as well as low isomerization.

More specifically, the inventors have discovered that formulations canbe prepared that will exhibit less than 8%, more typically less than 6%,even more typically less than 4%, and most typically less than 3% ofdegradation as determined by HPLC-UV, and that will exhibit less than10%, more typically less than 8%, even more typically less than 6%, andmost typically less than 4% of isomerization from R- to S-configurationas determined by HPLC-UV. Most notably, such formulations were found tobe stable over extended periods without antioxidants (e.g., at least 1month, or at least two months, or at least three months), even atelevated storage temperatures (e.g., accelerated storage conditions suchas 40° C. and 75% relative humidity (+/−5%)). Even more remarkable, suchformulations could also be subjected to thermal sterilization, andparticularly sterilizing to sterility (e.g., over at least 5 min, or atleast 10 min, or at least 15 min at 121° C.), without substantialincrease (i.e., >1.5%, or >1.0%, or >0.7%) of the S-isomer ofnorepinephrine.

Additionally, it should be appreciated that contemplated formulationscan be filled in a polymer bag (e.g., polypropylene) or other containerthat may subsequently be placed into a secondary container together withan oxygen scavenger, and especially a metal-free oxygen scavenger. Mosttypically, at least one of the polymer bag and the secondary containermay be impervious to light in general or light of a wavelength thatpromotes photo-initiated degradation. For example, containers may bemetalized (e.g., aluminized) or combined or coated with carbonaceousmaterials or other dye(s). If desired, contemplated formulations aresufficiently stable to also allow filling into containers using ablow-fill-seal (BFS) process.

Therefore, contemplated norepinephrine formulations of the inventivesubject matter can advantageously be provided in a ready-to-inject formto thereby avoid the inconvenience associated with diluting concentratedsmall volume norepinephrine parenteral formulations into diluents priorto infusion. Thus, the ready-to-inject formulations also eliminatemicrobial contamination risks and calculation errors associated withdilution. Most typically, contemplated formulations will be available ina range of concentrations commonly required by medical practitioners foremergency restoration of blood pressure, for example in cases of acutehypotension. Consequently, norepinephrine will typically be present informulations at a concentration of between 10 μg/ml and 100 μg/ml,including concentration of 16 μg/ml (+/−10%), 32 μg/ml (+/−10%), and 64μg/ml (+/−10%).

As will be readily appreciated, the norepinephrine for preparation ofcontemplated formulations is preferably (R)-Norepinephrine, orenantiomerically pure (i.e., at least 98% R-isomer) norepinephrine.However, in less preferred aspects, isomeric purity can also be between95-98%, or even between 90-95%. Of course, it should also be appreciatedthat the norepinephrine may be a salt of any suitable andpharmaceutically acceptable form, including mineral salts (e.g., HClsalt) and organic salts (e.g., bitartrate). Similarly, where desired,the norepinephrine may also be used in any suitable prodrug form (e.g.,β,3-dihydroxytyrosine, L-dihydroxyphenylserine, etc.).

Suitable buffers are generally buffers that stabilize the pH of thecontemplated liquid formulations in an acidic pH range and willtherefore include glycine buffers, citrate buffers, citrate/phosphatebuffers, acetate buffers, etc. However, the inventors have furtherdiscovered that where the norepinephrine is provided as thenorepinephrine bitartrate salt, a buffer can advantageously be omittedand the pH can be adjusted with suitable acid and/or base as is wellknown in the art. Notably, the bitartrate appeared to act as a weakbuffer in the stability range for the norepinephrine as is shown in moredetail below. Most typically the pH of the formulation will be less than5.0 and more typically less than 4.5, and most typically less than 4.3,but higher than 3.0, more typically higher than 3.5, and most typicallyhigher than 3.7. For example, suitable buffers will have a pH in therange of between 3.7 and 4.3, or between 3.7 and 4.0, or between 3.8 and4.1, or between 3.9 and 4.2, or between 4.0 and 4.2. Notably, such pHrange provided remarkable stability for low concentrations ofnorepinephrine, especially when in combination with a chelator and asalt. While not limiting to the inventive subject matter, the bufferstrength is typically relatively low, for example, equal or less than100 mM, and more typically equal or less than 50 mM, and most typicallybetween 5 mM and 20 mM (e.g., 10 mM).

Moreover, in further contemplated aspects, the formulation will alsoinclude one or more chelating agents, and particularly metal ionchelators. For example, suitable chelators include various bicarboxylicacids, tricarboxylic acids, and aminopolycarboxylic acids such asethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), andpenta(carboxymethyl)diethylenetriamine (DTPA), and salts and hydratesthereof. While not limiting to the inventive subject matter, it iscontemplated that the metal ion chelators will slow down both thebaseline and metal ion-stimulated autoxidation of norepinephrine.Remarkably, the inventors unexpectedly observed that the desirableeffect of the chelators was observable at relatively low concentrationsof the chelators. For example, reduction of the baseline and metalion-stimulated autoxidation of norepinephrine was observed at chelatorconcentrations of between 1 μg/ml and 10 μg/ml, and between 10 μg/ml and100 μg/ml. Interestingly, the chelators, and especially theaminopolycarboxylic acids retained stabilizing effect despite therelatively low pH favoring protonated forms of the chelators.

With respect to suitable salts it is contemplated that the salt is apharmaceutically acceptable salt that can be used to increase tonicity.Therefore, pharmaceutically acceptable salts are contemplated, andespecially NaCl, at a concentration of at least 0.6 wt %, or at least0.7 wt %, or at least 0.8 wt %, or at least 0.9 wt %. For example,suitable salt concentrations are between 0.6 wt % and 1.2 wt %.Depending on the particular salt concentration, additional tonicityagents may be added and suitable tonicity agents include glycerol,thioglycerol, mannitol, lactose, and dextrose. The amount of tonicityadjusting agent used can be adjusted to obtain osmolality of theformulations in the range of 260 to 340 mOsm/kg. An osmometer can beused to check and adjust the amount of tonicity adjusting agent to beadded to obtain the desired osmolality.

It should further be appreciated that contemplated compositions aresubstantially free of antioxidants (i.e., do not include antioxidants inan amount effective to reduce degradation of total norepinephrine by atleast 1% when stored over a period of at least three months at 25° C.Indeed, the inventors unexpectedly discovered that some formulationswith antioxidants (particularly with ascorbic acid) had decreasedstability. Notably, contemplated formulations were stable as describedin more detail below, even in the absence of effective quantities ofantioxidants, especially where deoxygenated solvents (e.g., typicallywater and/or buffer) were employed. Deoxygenation (i.e., reduction ofmolecular dissolved oxygen) can be achieved in numerous manners,including sparging with inert gases (e.g., helium, various freons,argon, xenon), agitation under vacuum, and/or using enzymatic systemsthat deplete a solution of dissolved oxygen (see e.g., U.S. Pat. No.9,187,779). Additionally, or alternatively, ingress of molecular oxygeninto the formulation can also be reduced by co-packaging a containerwith the formulation in a secondary container that includes an oxygenscavenger, and especially a metal-free oxygen scavenger (e.g., GLS100,Ageless®, Pharmakeep®, all commercially available from Mitsubishi GasChemical America).

With respect to the sterilization of contemplated formulations it shouldbe appreciated that contemplated formulations may be sterilized usingall known manners of sterilization, including filtration through 0.22micron filters, heat sterilization, autoclaving, radiation (e.g., gamma,electron beam, microwave). Unexpectedly, and as shown in more detailbelow, the inventors have also discovered that contemplated formulationswere heat stable and did not undergo significant isomerization, evenunder conditions of sterilization (exposure to high-pressure saturatedsteam) at 121° C. for at least 5, or at least 10, or at least 15minutes.

Based on the unexpected heat stability, the formulations contemplatedherein can also be filtered through a 0.22 micron filter, and filled into a polyethylene, polypropylene or low-density polyethylene containersin a blow-fill-seal (BFS) process. BFS is a form of advanced asepticmanufacturing wherein the container is formed, filled, and sealed in onecontinuous, automated system not requiring human intervention. Theprocess begins with the extrusion of plastic granules in the form of ahot hollow pipe of molten plastic called a parison. The next step is theblow molding of the container with an open top through which thecontainer is filled, all while the plastic remains hot and in a moltenstate. Once filled, the container is hermetically sealed and cooled. Theblow-fill seal process can take several seconds, and contemplatedready-to-inject compositions advantageously are formulated to withstandthe temperature and pressure requirements without substantialdegradation of norepinephrine (e.g., less than 5 wt %, less than 3 wt %,less than 2 wt %, less than 1 wt % degradation).

Once the norepinephrine formulations are filled in large volumepolymeric, semi-permeable infusion containers (e.g., BFS container orflexible IV bags), the containers can optionally be layered or coveredwith a secondary packaging system including an aluminum pouch or otheroxygen scavenger. For example, the BFS containers can further be sealedin an oxygen and moisture barrier blister packaging. The blisterpackaging can comprise one or more layers, and the one or more layerscan include aluminum foil or other oxygen absorber having an OxygenTransmission Rate (OTR) between 0.0005 to 5.00 cc/100 in²/24 hrs.Additionally or alternatively, one or more oxygen absorbers (metal ormetal free, organic material) can be incorporated into any portion ofthe BFS container, the secondary packaging system, or between the two(e.g., between the BFS container and the multi-layer packaging) suchthat the oxygen absorber removes at least a portion of oxygen from theair surrounding said oxygen-sensitive drug. A beneficial feature of theoxygen absorber is the absorbance and removal of oxygen present in theprimary packaging and in the liquid drug itself. Notably, it was foundthat the oxygen absorber also removed residual headspace oxygen in theprimary packaging and also dissolved oxygen in the liquid over time,thereby further improving stability of norepinephrine.

The following examples are provided for illustrative purposes only andshould not be interpreted as limiting the present invention.

Examples

The following examples illustrate some of the experiments leading to theformulations according to the inventive subject matter, however, shouldnot be construed to limit the scope of the claims in any way.

Stability and Isomerization: The ionization behavior of norepinephrinein aqueous solution is complex. Common with other o-hydroquinonesystems, norepinephrine in aqueous solution is susceptible to oxidationto form the corresponding o-quinone, which can then also undergo varioussecondary reactions, which also becomes more prevalent as the pH becomesmore alkaline. Norepinephrine may further isomerize to thepharmacologically less active S-enantiomer at low pH values,corresponding to protonation of the hydroxyl group at the benzylicchiral center. Therefore, to prevent norepinephrine cyclizationreactions pH values less than 6.0 are desired. A pH range of 3.0 to 6.2was screened to determine pH of optimum stability. Composition ofnorepinephrine bitartrate equivalent to 16 μg/mL norepinephrine base atvarious pH values were prepared are described below, with Table 4listing compositions of norepinephrine bitartrate in citrate buffer (10mM),

For preparation of the solutions, about 90% of the final quantity ofwater was collected in a glass media bottle. Nitrogen (N₂) gas waspurged for about thirty minutes to reduce the dissolved oxygen levels.Sodium chloride was added and the solution was stirred until ahomogenous solution was obtained. Citric acid was added and the solutionwas stirred until a homogenous solution was obtained. The pH of the bulksolutions was adjusted to pH 3.0, 3.4, 3.8, 4.2, 4.6, 5.0, 5.4, 5.8, and6.2, respectively for each formulation composition using sufficientquantity of 10% w/v sodium hydroxide or 10% w/v hydrochloric acid.Norepinephrine bitartrate was added and the solution was stirred forapproximately 10 minutes until a clear solution was formed. Solutionswere made up to volume with water. The solutions were filled into 10 mLglass vials, overlaid with nitrogen, stoppered, and sealed. Thestability was studied at 4° C., 25° C., and 60° C. by assay. Sampleswere observed visually for precipitation and change in color for aperiod of 7 days. Data are presented in Table 5.

TABLE 4 Compositions of Norepinephrine Bitartrate for pH dependentstability in Citrate Buffer (10 mM). Concentration (mg/mL) Ingredients III III IV V VI VII VIII IX Norepinephrine Bitartarate 0.016 0.016 0.0160.016 0.016 0.016 0.016 0.016 0.016 equivalent to Norepinephrine baseSodium Chloride 9 9 9 9 9 9 9 9 9 Citric acid 1.92 1.92 1.92 1.92 1.921.92 1.92 1.92 1.92 Sodium Citrate 2.94 2.94 2.94 2.94 2.94 2.94 2.942.94 2.94 HCl/NaOH (q.s. pH) 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2 Waterfor Injection (q.s. mL) 1 1 1 1 1 1 1 1 1

TABLE 5 Effect of pH on stability of Norepinephrine Bitartrate incitrate buffer. Assay Assay Temperature Formulation To T₇ pH ColorPrecipitation  4° C. I 96.4 96.5 3.0 No No II 98.0 97.5 3.4 No No III99.0 98.5 3.8 No No IV 99.1 98.4 4.2 No No V 98.1 98.6 4.6 No No VI 98.498.1 5.0 No No VII 97.1 96.6 5.4 No No VIII 97.8 97.5 5.8 No No IX 91.591.2 6.2 No No 25° C. I 96.4 96.4 3.0 No No II 98.0 97.5 3.4 No No III99.0 97.9 3.8 No No IV 99.1 97.7 4.2 No No V 98.1 97.3 4.6 No No VI 98.497.3 5.0 No No VII 97.1 95.9 5.4 No No VIII 97.8 94.5 5.8 No No IX 91.580.4 6.2 Yes No 60° C. I 96.4 95.2 3.0 No No II 98.0 95.0 3.4 No No III99.0 95.2 3.8 No No IV 99.1 93.2 4.2 No No V 98.1 88.9 4.6 No No VI 98.477.4 5.0 Yes No VII 97.1 46.8 5.4 Yes No VIII 97.8 NT 5.8 Yes No IX 91.5NT 6.2 Yes No

No change in physical appearance was observed in the solutions stored at4° C. In the solutions stored at 25° C., a change in color was observedat pH 6.2. Red brown color was observed in solutions stored at or abovepH 5.0 at 60° C. Oxidation and color formation are very common withnorepinephrine in unfavorable conditions and the speed of the reactionand the nature of the final products are dependent on the catalysts(e.g., metal ion impurities) and buffers employed. A pH range from 3.0to 4.5 was selected for further testing.

Stability of Norepinephrine in selected pH ranges and formulations: Theformulations for the next experiments are shown in Table 6 below,involving three different compositions of norepinephrine bitartrate atthree different pH (3.5, 4.0, 4.5, and 5.0) values. Lab scale batcheswere prepared and subjected to lab scale stability tests at accelerated(40° C./75% RH) and long term stability (25° C./60% RH) storageconditions. The test results from the stability studies are presented inTable 7-Table 10, with CCS indicating Clear colorless solution; NDindicating Not Detected; NR indicating Not Reported (<0.05%); and NTindicating Not Tested.

TABLE 6 Formulation composition selected for further developmentactivities and optimization Quantity (mg/mL) Formulation Ingredient X XIXII XIII Norepinephrine Bitartrate 0.016 0.016 0.016 0.016 EdetateSodium 0.10 0.10 0.10 0.10 Sodium chloride 9 9 9 9 HCl/NaOH q.s. q.s.q.s. q.s. pH 3.5 pH 4.0 pH 4.5 pH 5.0 Water for Injection Q.S. 1 ml 1 ml1 ml 1 ml Dissolved Oxygen (ppm) <1 <1 <1 <1

TABLE 7 Stability study of Formulation X - Norepinephrine BitartrateInjection (16 μg/ml) filled in glass vial (pH 3.5). Storage Condition 25± 2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point 1 2 3 4 5 6 1 2 3 45 6 Initial Month Month Month Month Month Month Month Month Month MonthMonth Month Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS CCSCCS pH 3.50 3.65 3.59 3.56 3.58 3.54 3.48 3.66 3.61 3.59 3.64 3.60 3.59Assay 101.4 99.6 97.1 97.1 101.0 102.3 102.2 99.5 97.0 98.7 100.4 101.7101.4 S-form NT NT NT NT 1.8 2.2 2.2 NT NT NT 7.6 8.1 9.8 Total ND ND NDND ND ND ND ND ND ND ND ND ND

TABLE 8 Stability study of Formulation XI- Norepinephrine BitartrateInjection (16 μg/ml) filled in glass vial (pH 4.0). Storage Condition 25± 2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2Month 3 Month 6 Month 1 Month 2 Month 3 Month 6 Month Appearance CCS CCSCCS CCS CCS CCS CCS CCS CCS pH 4.02 3.96 3.98 3.97 3.91 4.01 3.99 4.024.03 Assay 101.3 98.7 95.5 99.2 100.5 98.6 95.3 97.1 97.5 S-form NT NTNT NT 1.7 NT NT NT 7.8 Total Impurities 0.1 ND 0.06 ND 0.80 ND 0.06 0.10.79

TABLE 9 Stability study of Formulation XII - Norepinephrine BitartrateInjection (16 μg/ml) filled in glass vial (pH 4.5). Storage Condition 25± 2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2Month 3 Month 6 Month 1 Month 2 Month 3 Month 6 Month Appearance CCS CCSCCS CCS CCS CCS CCS CCS CCS pH 4.50 4.35 4.36 4.32 4.33 4.33 4.40 4.394.29 Assay 100.1 98.9 95.5 98.2 97.9 97.1 92.5 93.7 77.2 S-form NT NT NTNT 1.2 NT NT NT 2.9 Total Impurities ND 0.32 0.79 0.52 3.41 1.18 0.385.59 10.38

TABLE 10 Stability study of Formulation XIII - Norepinephrine BitartrateInjection (16 μg/ml) filled in glass vial (pH 5.0). Storage Condition 25± 2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2Month 3 Month 4 Month 1 Month 2 Month 3 Month 4 Month Appearance CCS CCSCCS CCS CCS CCS CCS CCS CCS pH 4.99 4.62 4.51 4.57 4.51 4.87 4.81 4.834.53 Assay 102.7 100.5 95.6 99.2 100.4 98.3 89.8 87.0 72.3 S-form NT NTNT NT 1.2 NT NT NT 3.0 Total Impurities ND 0.75 0.81 0.48 1.29 0.94 2.45.39 14.91

Based on the above considerations, the effect of different levels ofEDTA on stability of norepinephrine was determined. Three batches atconcentrations of 16 μg/mL, 32 μg/mL, and 64 μg/mL were made with EDTAconcentrations of 100 μg/mL: Formulation XIV (16 μg/mL), Formulation XV(32 μg/mL), Formulation XVI (64 μg/mL). Two additional batches were madeat 10 μg/mL EDTA Formulation XVII and 1 μg/mL EDTA (Formulation XVIII)at 64 μg/mL Norepinephrine. The composition of the batches is specifiedin Table 11. The drug product was compounded as described earlier andpackaged in 250 mL in polypropylene bags. This was further packed intoaluminum overwrap with an oxygen scavenger (GLS 100, Mitsubishi GasChemicals). The batches were then stored at room temperature andaccelerated temperature conditions.

TABLE 11 Formulation composition selected with different level of EDTAconcentrations. Quantity (mg/mL) Formulation Number Ingredient XIV XVXVI XVII XVIII Norepinephrine Bitartrate 0.016 0.032 0.064 0.064 0.064Edetate Sodium 0.10 0.10 0.10 0.010 0.0010 Sodium chloride 9 9 9 9 9Hydrochloric Acid/ q.s. q.s. q.s. q.s. q.s. Sodium Hydroxide pH 4.0 pH4.0 pH 4.0 pH 4.0 pH 4.0 Water for Injection q.s. q.s. q.s. q.s. q.s. 1ml 1 ml 1 ml 1 ml 1 ml

The resultant stability data on these formulations are presented inTable 12-Table 16 (CCS—Clear colorless solution; ND—Not Detected). Theresults of the stability studies at different amounts of EDTA at pH 4.0indicates that both 0.01%, 0.001% of EDTA significantly prevented thedegradation rate of norepinephrine in terms of known and unknownimpurities. Moreover, with respect to isomerization from the R-isomer tothe S-isomer it was notably observed that the amount of EDTA hadsubstantially no influence on racemization or enantiomer formationduring stability and after autoclaving.

TABLE 12 Stability study of Formulation XIV - Norepinephrine bitartrateinjection (16 μg/mL); pH 4.0 at 100 μg/mL EDTA. Storage Condition 25 ±2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2Month 3 Month 1 Month 2 Month 3 Month Appearance CCS CCS CCS CCS CCS CCSCCS pH 3.99 3.96 4.08 4.08 4.02 4.08 4.08 Assay 98.5 100.4 100.1 99.7100.3 100.0 99.5 S-form 0.9 1.1 1.4 1.3 1.9 2.9 4.2 Total Impurities0.05 ND ND ND ND 0.10 0.38

TABLE 13 Stability study of Formulation XV - Norepinephrine bitartrateinjection (32 μg/mL); pH 4.0 at 100 μg/mL EDTA. Storage Condition 25 ±2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2Month 3 Month 1 Month 2 Month 3 Month Appearance CCS CCS CCS CCS CCS CCSCCS pH 4.01 3.99 4.08 4..08 4.02 4.08 4.08 Assay 101.0 102.9 97.1 100.7102.9 99.4 100.6 S-form 0.9 1.1 1.3 1.4 1.9 3.0 4.1 Total Impurities0.06 ND ND ND ND ND 0.14

TABLE 14 Stability study of Formulation XVI - Norepinephrine bitartrateinjection (64 μg/mL); pH 4.0 at 100 μg/mL EDTA. Storage Condition 25 ±2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2Month 3 Month 1 Month 2 Month 3 Month Appearance CCS CCS CCS CCS CCS CCSCCS pH 4.00 3.99 4.08 4.08 3.98 4.07 4.07 Assay 98.4 103.2 98.7 100.2104.6 99.3 99.8 S-form 0.9 1.1 1.3 1.3 2.0 3.2 4.2 Total Impurities 0.06ND 0.12 ND ND ND ND

TABLE 15 Stability study of Formulation XVII - Norepinephrine bitartrateinjection (64 μg/mL); pH 4.0 at 10 μg/mL EDTA. Storage Condition 25 ± 2°C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial 1 Month 2 Month 3Month 1 Month 2 Month 3 Month Appearance CCS CCS CCS CCS CCS CCS CCS pH4.00 3.98 4.06 4.06 3.99 4.05 4.05 Assay 102.7 105.7 103.4 104.3 107.8103.6 103.9 S-form 0.9 1.1 1.2 1.5 2.0 3.3 4.3 Total 0.06 ND ND ND ND0.26 ND

TABLE 16 Stability study of Formulation XVIII - Norepinephrinebitartrate injection (64 μg/mL); pH 4.0 at 1 μg/mL EDTA. StorageCondition 25 ± 2° C./60 ± 5% RH 40 ± 2° C./75 ± 5% RH Time Point Initial1 Month 2 Month 3 Month 1 Month 2 Month 3 Month Appearance CCS CCS CCSCCS CCS CCS CCS pH 4.00 3.98 4.07 4.07 4.02 4.06 4.06 Assay 98.7 102.6100.4 100.4 105.0 99.9 99.2 S-form 0.9 1.1 1.3 1.4 2.0 3.2 4.3 Total0.06 ND ND ND ND ND ND

Sterilization and Stability: The volume for ready-to-inject formulationsis 250 mL and as such classifies as a large volume parenteral (LVP). Toachieve a desired or required sterility assurance level of 10⁻⁶ for aLVP terminal sterilization via heat it is typically required. Theinventors therefore investigated whether or not contemplatedformulations could be terminally sterilized via autoclaving.

Formulations at a concentration 16 μg/mL and 64 μg/mL (Formulation XVII)Norepinephrine base were prepared substantially as shown in Table 11above and packaged in secondary packaging of aluminum overwrap with anoxygen scavenger and shipped for terminal sterilization. The secondarypackaging was removed and the bags were terminally sterilized usingsteam sterilizer (Fedegari, Model # FOB3) with an air over-pressure(AOP) sterilization cycle. The terminal sterilization was performed at121° C. for 5, 10, and 15 min. Post completion of sterilizationtemperature, the bags underwent spontaneous cooling to 95° C. and forcedcooling to 70° C. The total exposure time and calculated F₀ values were11.09, 17.04, and 22.42 for 5 min, 10 min, and 15 min cyclesrespectively. The bags were then analyzed for assay, impurities, andS-isoform, and the results are shown in Table 17 and Table 18.

TABLE 17 Stability study of Norepinephrine bitartrate injection (16μg/mL) filled in 100 mL PP bags (pH 4.0); 10 μg/mL EDTA; terminallysterilized. Time Point Initial 5 Min 10 Min 15 Min Bag Number 1 2 3 1 23 1 2 3 Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS pH 3.76 3.853.78 3.77 3.76 3.76 3.78 3.76 3.75 3.76 Dissolved Oxygen 0.63 4.93 4.864.89 0.75 0.48 0.55 0.65 0.78 0.77 Assay 103.1 103.1 103.1 103.1 103.1103.0 103.1 103.1 103.2 103.1 S-Form 1.0 3.0 3.0 3.0 3.8 3.7 3.7 4.3 4.34.3 Total ND ND ND ND ND ND ND ND ND ND

TABLE 18 Stability study of Norepinephrine bitartrate injection (64μg/ml) filled in 100 mL PP bags (pH 4.0); 10 μg/mL EDTA terminallysterilized. Time Point Initial 5 Min 10 Min 15 Min Bag Number 1 2 3 1 23 1 2 3 Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS pH 3.74 3.743.75 3.73 3.74 3.74 3.76 3.74 3.73 3.74 Dissolved Oxygen 0.69 5.15 5.035.00 0.52 0.59 0.75 0.69 0.80 0.74 Assay 101.2 102.2 101.2 101.5 101.7101.2 101.3 101.2 101.3 102.2 S-Form 1.0 3.0 3.0 3.0 3.7 3.7 3.7 4.3 4.34.3 Total ND ND ND ND ND ND ND 0.1 ND ND

As can be seen from the data, the S-isoform appears to increaseproportionally to time during the terminal sterilization cycle. Noincrease in reportable impurities was observed.

Test method—Determination of norepinephrine and degradation products:Separation of Norepinephrine and related compounds was performed using agradient HPLC method with UV detection. Pentofluorophenylpropylterminated silica was used as a stationary phase for chromatographicanalysis. The mobile phase was prepared by mixing water and methanol,with both solvents containing formic acid. Related compounds weredefined by their relative retention times (RRT) based on the NE peakretention time. Quantitation of related compounds was accomplished bycomparing the corresponding peak area from a sample solutionchromatogram to that of the NE peak from a Reference Standard (RS)solution of a known concentration. Relative Response Factors (RRF) wereused to correct for chemical structure effects on the responses of theidentified impurities. Chromatography was performed using parameters andmethods as shown in Table 19.

TABLE 19 HPLC Waters Alliance e2695 Column Supelco Discovery HS F-5Column, 3 μm, 4.6 × 150 mm Column Temperature 35 ° C. Sample TemperatureAmbient Injection volume 85.0 μL Flow Rate 0.8 mL/min DetectionSpectrum: 200-600 nm, resolution 1.2 nm Single channel: 280 nm,resolution 4.8 nm PDA Filter Time Constant: Normal Sampling rate: 5points/sec Solution A 0.1% Formic acid in Water Solution B 0.1% Formicacid in Methanol Time (mins) % Solution A % Solution B Mobile Phase 0100 0 3 100 0 6 93 7 8 93 7 15 88 12 30 2 98 35 2 98 36 100 0 40 100 0

Test Method—Identification, Assay and Enantiomeric Purity ofNorepinephrine: Identification and quantification of S-norepinephrineand R-norepinephrine was performed using an HPLC method with UVdetection. HPLC-UV was used to separate and quantitate the amount of(R)- and (S)-enantiomers of norepinephrine (NE) present in the drugproduct with the NE concentrations of 16, 32 and 64 μg/ml. Thecomparison of the sum of (R)- and (S)-peak responses in a samplechromatogram versus a reference standard chromatogram gives the totalamount of NE. The (S)-enantiomer was quantitated based on its peakresponse as the percentage of the total peak response of bothenantiomers.

More specifically, determination of R- and S-enantiomers ofnorepinephrine in the drug product solution was performed using anisocratic reverse-phase HPLC method with UV detection. Separation wasachieved by using a protein-based column with functional chiralselectors. The chiral selector is cellobiohydrolase (CBH), a stableenzyme that has been immobilized onto spherical silica particles. Thisenzyme preferentially separates compounds containing one or more basicnitrogen groups together with one or more hydrogen-accepting orhydrogen-donating groups. Chromatography was performed using parametersand methods as shown in Table 20.

TABLE 20 HPLC Agilent 1260 Infinity Column Daicel Chiralpak CBH ™column, 5 μm, 4.0 × 100 mm Column 27° C. ± 2° C. Temperature SampleAmbient Temperature Injection 20.0 μL for 16 mcg/mL, volume 10.0 μL for32 mcg/mL, 5.0 μL for 64 mcg/mL Flow Rate 0.9 mL/min Dection Singlechannel: 280 nm, resolution 4.8 nm Spectrum: 200-600 nm, resolution 1.2nm Mobile phase: Buffer/IPA 95:5 v/v Buffer: Sodium Phosphate, DisodiumEdetate, pH 6.0 Run Time 8 min

While contemplated formulations can be administered following variousprotocols, the inventors contemplate that administration of theformulations, especially administration for treatment of hypotension,will follow a protocol that comprises at least two distinct steps, withan accelerated administration followed by a maintenance administrationas exemplarily described in Table 21 below.

TABLE 21 Initial Dose Maintenance Dose Presen- Concen- Dose per FlowDose per Flow tation tration Minute Rate Minute Rate (mg/mL) (μg/mL)(μg/min) (mL/min) (μg/min) (mL/min) 16 μg/mL 16 8-12 0.500- 2-4 0.125-(4 mg in 0.750 0.250 250 mL) 32 μg/mL 32 0.250- 0.062- (8 mg in 0.3750.125 250 mL) 64 μg/mL 64 0.125- 0.031- (16 mg in 0.187 0.062 250 mL)

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

It should be apparent, however, to those skilled in the art that manymore modifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of thedisclosure. One skilled in the art will recognize many methods andmaterials similar or equivalent to those described herein, which couldbe used in the practice of the present invention. Indeed, the presentinvention is in no way limited to the methods and materials described.

Moreover, in interpreting the disclosure all terms should be interpretedin the broadest possible manner consistent with the context. Inparticular the terms “comprises” and “comprising” should be interpretedas referring to the elements, components, or steps in a non-exclusivemanner, indicating that the referenced elements, components, or stepscan be present, or utilized, or combined with other elements,components, or steps that are not expressly referenced.

What is claimed is:
 1. A ready-to-inject norepinephrine composition,consisting essentially of: an aqueous solution comprising a chelatingagent and a pharmaceutically acceptable salt; wherein the chelatingagent is an aminopolycarboxylic acid present in an amount of equal orless than 100 μg/ml, and wherein the pharmaceutically acceptable salt ispresent in an amount of at least 0.7 wt %; norepinephrine bitartratedissolved at a concentration of between 16 and 64 μg/ml, and wherein thenorepinephrine is an R-isomer; and wherein the ready-to-administernorepinephrine composition is formulated to have a pH range of between3.8 and 4.2 and has a composition such that after storage over at leastthree months equal or less than 10% of the R-isomer form will isomerizeto the S-isomer and such that equal or less than 5% of the totalnorepinephrine will degrade to degradation products.
 2. The compositionof claim 1 wherein the ready-to-administer norepinephrine compositionhas a pH between 3.8 and 4.1.
 3. The composition of claim 1 wherein theready-to-administer norepinephrine composition has a pH between 3.9 and4.2.
 4. The composition of claim 1 wherein the ready-to-administernorepinephrine composition has a pH of 4.0.
 5. The composition of claim1 wherein the ready-to-administer norepinephrine composition issubstantially free of sodium metabisulfite.
 6. The composition of claim1 wherein the aqueous solution is water.
 7. The composition of claim 1wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).8. The composition of claim 1 wherein the chelating agent is present ata concentration of between 10 μg/ml and 100 μg/ml.
 9. The composition ofclaim 1 wherein the chelating agent is 10 μg/ml.
 10. The composition ofclaim 1 wherein the aqueous solution is water, wherein the chelatingagent is ethylenediaminetetraacetic acid (EDTA) at a concentration ofbetween 10 μg/ml and 100 μg/ml, and wherein the ready-to-administernorepinephrine composition is substantially free of sodiummetabisulfite.
 11. The composition of claim 1 wherein thepharmaceutically acceptable salt is present in an amount of 0.9 wt %.12. The composition of claim 10 wherein the pharmaceutically acceptablesalt is present in an amount of 0.9 wt %.
 13. The composition of claim 1wherein the norepinephrine is present at a concentration of 16 μg/ml(+/−10%), 32 μg/ml (+/−10%), or 64 μg/ml (+/−10%).
 14. The compositionof claim 10 wherein the norepinephrine is present at a concentration of16 μg/ml (+/−10%), 32 μg/ml (+/−10%), or 64 μg/ml (+/−10%).
 15. Thecomposition of claim 12 wherein the norepinephrine is present at aconcentration of 16 μg/ml (+/−10%), 32 μg/ml (+/−10%), or 64 μg/ml(+/−10%).
 16. The composition of claim 1 wherein the storage over atleast three months is at 40° C. and 75% (+/−5%) relative humidity. 17.The composition of claim 1 wherein equal or less than 6% of the R-isomerform will isomerize to the S-isomer.
 18. The composition of claim 10wherein equal or less than 6% of the R-isomer form will isomerize to theS-isomer.
 19. The composition of claim 1 wherein equal or less than 3.5%of the total norepinephrine will degrade to degradation products. 20.The composition of claim 1 wherein the composition has a dissolvedoxygen concentration of equal or less than 1 ppm.